Media used in this study consisted of 1% w/v yeast extract, 2% w/v peptone, and 2% w/v glucose (YPD). Synthetic complete (SC) media consisted of 6.7 g/L Yeast Nitrogen Base with ammonium sulfate and without amino acids, 1.77 g/L CSM-Ura (Formedium), 50 mg/L uracil (Sigma), and either 2% w/v glucose (SC-Glu), 2% w/v raffinose (SC-Raf) or 2% w/v galactose + 2% raffinose w/v (SC-Gal). YPD and SC media containing Hygromycin B (Invitrogen) (200 mg/L), or Nourseothricin (clonNAT) (100 mg/L) were used for the selection of yeast transformants and plasmid maintenance. Plates of these media were made with 2% agar. Canavanine plates consisted of 6.7 g/L Yeast Nitrogen Base with ammonium sulfate and without amino acids, 0.74 g/L CSM-Arg (Formedium), 60 mg/liter l -canavanine (Sigma-Aldrich), 2% w/v agar, and 2% w/v glucose.
Yeast nitrogen base
Manufactured by Formedium
Sourced in United Kingdom
Yeast nitrogen base is a defined growth medium used for cultivating microorganisms, particularly yeast. It provides essential nitrogen sources, vitamins, and minerals required for microbial growth and metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Glucose, by Merck Group (6 mentions)
D glucose, by Carl Roth (3 mentions)
Glucose, by Formedium (3 mentions)
Hygromycin b, by Thermo Fisher Scientific (3 mentions)
Methotrexate, by Thermo Fisher Scientific (2 mentions)
Synthetic complete, by Formedium (2 mentions)
Geneticin, by Formedium (2 mentions)
Peptone, by BD (2 mentions)
Yeast nitrogen base, by Merck Group (2 mentions)
Yeast extract, by BD (2 mentions)
Csm trp 20 ade, by Formedium (2 mentions)
Uracil, by Merck Group (2 mentions)
L canavanine, by Merck Group (2 mentions)
Csm ura, by Formedium (2 mentions)
Csm arg, by Formedium (2 mentions)
Glucose, by Carl Roth (2 mentions)
Csm drop out complete, by Formedium (2 mentions)
L lysine, by Formedium (1 mentions)
Tryptone peptone, by Carl Roth (1 mentions)
Yeast extract, by Carl Roth (1 mentions)
28 protocols using yeast nitrogen base
1
Yeast Media and Antibiotic Selection
2
Comprehensive Yeast Strain and Plasmid Protocols
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A list of Saccharomyces cerevisiae strains and plasmids is provided in S4 Table . For strain maintenance and construction, strains were grown at 30°C under standard conditions. ncRNA single deletion strains used this study were taken from the ncRNA deletion collection created by Parker et al [10 (link), 78 (link)]. Deletion mutants were maintained on Yeast extract Peptone Dextrose Agar (YPDA) containing 200 μg/mL G418. Double deletion mutant strains were constructed by substituting the candidate SUT locus with the natNT2 cassette and were maintained on YPDA containing 100 μg/mL clonNAT.
For construction of strains ectopically expressing particular SUTs, isogenic wild-type and ncRNA deletion mutant strains cells were transformed with pRS416-Gal1-Cyc1 overexpression plasmid containing the ncRNA of interest. Resulting strains were maintained in a synthetic minimal media lacking uracil (SD-Ura: 1X Yeast Nitrogen Base (YNB) (Formedium); 1X Complete Supplement Mixture (CSM)–Ura (Formedium); 2% (w/v) glucose). For phenotypic rescue studies, strains were grown to an optical density at 600 nm (OD600) of 0.5 in YP (1% yeast extract, 2% peptone) medium supplemented with 2% raffinose (YPRaf) at 30°C and induced with YP medium containing 2% galactose (YPGal) for 2 hours before being harvested for spot test assays.
For construction of strains ectopically expressing particular SUTs, isogenic wild-type and ncRNA deletion mutant strains cells were transformed with pRS416-Gal1-Cyc1 overexpression plasmid containing the ncRNA of interest. Resulting strains were maintained in a synthetic minimal media lacking uracil (SD-Ura: 1X Yeast Nitrogen Base (YNB) (Formedium); 1X Complete Supplement Mixture (CSM)–Ura (Formedium); 2% (w/v) glucose). For phenotypic rescue studies, strains were grown to an optical density at 600 nm (OD600) of 0.5 in YP (1% yeast extract, 2% peptone) medium supplemented with 2% raffinose (YPRaf) at 30°C and induced with YP medium containing 2% galactose (YPGal) for 2 hours before being harvested for spot test assays.
3
Yeast Cell Culture and Preparation
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All chemicals were purchased from Sigma‐Aldrich (Darmstadt, Germany), unless otherwise indicated. Cells were cultured at 30 °C with 200 RPM shaking in 50 mL CELLreactor™ filter top tubes (Greiner Bio‐On, Kremsmünster, Austria). Strains were grown overnight by inoculation in 5‐mL synthetic glucose media without uracil and lysine and supplemented with the dipeptide Lys‐Lys. Media were prepared by dissolving 2% w/v glucose and 0.69% w/v yeast nitrogen base (YNB) without amino acids (Formedium, Norfolk, UK). Media were supplemented with 0.19% w/v Kaiser synthetic mixture without uracil and lysine 51 , that is, a mixture containing 18 mg·L−1 adenine, 76 mg·L−1 myo‐inositol, 8 mg·L−1 para‐aminobenzoic acid, and 76 mg·L−1 of all 20 standard amino acids (l ‐leucine was added at 380 mg·L−1) except l ‐lysine (Formedium). Finally, 200 mg·L−1 Lys‐Lys was added. Subcultures were grown and diluted for two to three consecutive days such that the OD600 never exceeded 1. Cells were centrifuged at 3000 g for 5 min at 4 °C, supernatant was decanted, and cells were suspended in ice‐cold 100 mm potassium phosphate, 10 mm glucose, pH 6.0. This step was performed twice before suspension of the cells to an OD600 of 5.
4
Preparation of Standard Media Reagents
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All media were prepared according to standard protocols (Dymond, 2013 (link)). D (+)-Glucose (#HN06, tryptone/peptone (#8952), and yeast extract (#2363) were purchased from Carl Roth. Yeast nitrogen base (YNB) (#CYN0602), agar–agar (#AGA03), and complete supplement mixture (CSM complete) (#DCS0019) were purchased from FORMEDIUM.
5
Saccharomyces cerevisiae ncRNA Deletion Strains
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A list of Saccharomyces cerevisiae strains and plasmids is provided in S3 Table . For strain maintenance and construction, strains were grown at 30°C under standard conditions. ncRNA single deletion strains used this study were taken from the ncRNA deletion collection created by Parker, et al [10, 67] . Deletion mutants were maintained on Yeast extract Peptone Dextrose Agar (YPDA) containing 200 µg/mL G418. Double deletion mutant strains were constructed by substituting the candidate SUT locus with the natNT2 cassette and were maintained on YPDA containing 100 µg/mL clonNAT.
For construction of strains ectopically expressing particular SUTs, isogenic wild-type and ncRNA deletion mutant strains cells were transformed with pRS416-Gal1-Cyc1 overexpression plasmid containing the ncRNA of interest. Resulting strains were maintained in a synthetic minimal media lacking uracil (SD-Ura: 1X Yeast Nitrogen Base (YNB) (Formedium); 1X Complete Supplement Mixture (CSM) -Ura (Formedium); 2% (w/v) glucose). For phenotypic rescue studies, strains were grown to an optical density at 600 nm (OD 600 ) of 0.5 in YP (1% yeast extract, 2% peptone) medium supplemented with 2% raffinose (YPRaf) at 30°C and induced with YP medium containing 2% galactose (YPGal) for 2 hours before being harvested for spot test assays.
For construction of strains ectopically expressing particular SUTs, isogenic wild-type and ncRNA deletion mutant strains cells were transformed with pRS416-Gal1-Cyc1 overexpression plasmid containing the ncRNA of interest. Resulting strains were maintained in a synthetic minimal media lacking uracil (SD-Ura: 1X Yeast Nitrogen Base (YNB) (Formedium); 1X Complete Supplement Mixture (CSM) -Ura (Formedium); 2% (w/v) glucose). For phenotypic rescue studies, strains were grown to an optical density at 600 nm (OD 600 ) of 0.5 in YP (1% yeast extract, 2% peptone) medium supplemented with 2% raffinose (YPRaf) at 30°C and induced with YP medium containing 2% galactose (YPGal) for 2 hours before being harvested for spot test assays.
6
Yeast Growth and Starvation Conditions
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Yeast were grown in rich yeast extract peptone dextrose (YPD) media (2% glucose, 2% peptone, 1% yeast extract) or synthetic complete (SC) minimal medium (2% glucose, yeast nitrogen base supplemented with appropriate amino acid and base dropout mixtures; Formedium, Norfolk, UK) for maintaining of plasmids. Cells were routinely grown overnight to early / mid-log phase log phase (OD600 = <1.0) prior to experimental procedures, unless otherwise stated. For glucose starvtion experiments, rich and minimal media were supplemented with 2% raffinose instead of glucose but were otherwise identical. Geneticin (Formedium), used at a concentration of 250 μg/ml in rich media, and methotrexate (Alfa Aesar), used at a working concentration of 20 mM, was prepared in SC minimal media as described (MacDonald and Piper, 2015) . Expression of proteins from the CUP1 promoter was achieved by addition of 50 -100 µM CuCl2 to the media for at least 1 hour prior to downstream analysis.
7
Yeast Strain Engineering for Cellular Studies
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Strains used in this study are listed in Appendix Table S2 . Strains generated for screening procedures were in the S288C background. All other strains were in the W303 background. Chromosomal modifications were introduced using PCR products or linearized plasmids (Longtine et al, 1998 (link); Janke et al, 2004 (link)). The marker‐free strains SSY2836 and SSY2837 were derived from SSY2809 by transformation with the Pah1‐3HA or the pah1(7A)‐3HA sequence amplified from plasmids pSS1045 or pSS1047 followed by counterselection on 5‐fluoroorotic acid.
Strains were grown at 30°C on YPD, SCD‐MSG, or SCD medium as indicated. YPD medium consisted of 1% yeast extract (Becton Dickinson, Heidelberg, Germany), 2% peptone (Becton Dickinson), and 2% glucose (Merck, Darmstadt, Germany). SCD‐MSG medium consisted of 0.17% yeast nitrogen base without amino acids and ammonium sulfate (Formedium, Norfolk, UK), 0.1% monosodium glutamate, amino acids, and 2% glucose. SCD medium consisted of 0.7% yeast nitrogen base without amino acids (Sigma, Taufkirchen, Germany), amino acids, and 2% glucose.
Strains were grown at 30°C on YPD, SCD‐MSG, or SCD medium as indicated. YPD medium consisted of 1% yeast extract (Becton Dickinson, Heidelberg, Germany), 2% peptone (Becton Dickinson), and 2% glucose (Merck, Darmstadt, Germany). SCD‐MSG medium consisted of 0.17% yeast nitrogen base without amino acids and ammonium sulfate (Formedium, Norfolk, UK), 0.1% monosodium glutamate, amino acids, and 2% glucose. SCD medium consisted of 0.7% yeast nitrogen base without amino acids (Sigma, Taufkirchen, Germany), amino acids, and 2% glucose.
8
Yeast Strain Maintenance and Selection
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The S. cerevisiae strains used in this study are described in Table S5 in the supplemental material. Stocks were stored at −80°C in 15% glycerol. Strains that contained the URA3_CEN plasmid were grown on agar with 1 mg/mL of 5-fluoroorotic acid (5-FOA) (Kaixuan Chemical Co.) to select for uracil auxotrophs before construction of the query strains. Gene deletion libraries were maintained in synthetic complete (SC), synthetic dropout (SD), enriched sporulation, or yeast-peptone-dextrose agar as previously described (89 ). The media and solutions used included agar, amino acids, peptone, yeast extract, yeast nitrogen base (Formedium), ampicillin, atorvastatin calcium, glucose, monosodium glutamate, potassium acetate (Sigma-Aldrich), Geneticin sulfate, l -canavanine sulfate, S-aminoethyl-l -cysteine hydrochloride (thyalisine) (Carbosynth), nourseothricin sulfate (Werner BioAgents), and hygromycin B (Life Technologies). All antibiotics and supplement stocks were filter sterilized with 22-μm-pore filters (Jet Biofil).
9
Saccharomyces cerevisiae Oxidative Stress Assay
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The haploid Saccharomyces cerevisiae yeast strain BY4742 (Matα; his3Δ1;leu2Δ0, lys2Δ0, ura3Δ0) was used as a model fungus in tests with different thiosulfinates. The BY4742 mutant Δyap1 (YML007w) used in this study lacks a redox-sensitive transcription factor that is important for oxidative stress response. All mutants were obtained from the EUROSCARF Collection, University of Frankfurt (Main), Germany (http://www.euroscarf.de/ ).
Yeast was grown in CSM medium (0.79 g L−1 CSM Drop-Out: Complete [ForMedium, Norwich, United Kingdom]; 6.9 g/l Yeast Nitrogen Base [ForMedium, Norwich, United Kingdom]; 40 g L−1 D-Glucose [Carl Roth, Karlsruhe, Germany], 15 g L−1 agar for solid medium.
Yeast was grown in CSM medium (0.79 g L−1 CSM Drop-Out: Complete [ForMedium, Norwich, United Kingdom]; 6.9 g/l Yeast Nitrogen Base [ForMedium, Norwich, United Kingdom]; 40 g L−1 D-Glucose [Carl Roth, Karlsruhe, Germany], 15 g L−1 agar for solid medium.
10
Yeast Growth Conditions for Microscopy
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Yeast were grown in rich yeast extract peptone dextrose (YPD) medium (2% glucose, 2% peptone, 1% yeast extract) or synthetic complete (SC) minimal medium (2% glucose, yeast nitrogen base supplemented with appropriate amino acid and base dropout mixtures; Formedium, Norfolk, UK) for maintaining of plasmids. Cells were routinely grown overnight to early/mid-log phase (OD600≤1.0) prior to experimental procedures, unless otherwise stated. To minimise nutritional challenges prior to starvation experiments, very low density cultures (OD600=0.1) were adhered to concavalin A-treated coverslips for time-lapse microscopy. For glucose-starvation experiments, rich and minimal media were supplemented with 2% raffinose instead of glucose, but were otherwise identical. Geneticin (Formedium), used at a concentration of 250 μg/ml in rich media, and methotrexate (Alfa Aesar), used at a working concentration of 20 mM, were prepared in SC minimal medium as described previously (MacDonald and Piper, 2015 (link)). Expression of proteins from the CUP1 promoter was achieved by addition of 50–100 µM CuCl2 to the medium for at least 1 h prior to downstream analysis.
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